The innate immune system coordinates the inflammatory response to pathogens by a system that discriminates between self and non-self via receptors that identify classes of molecules synthesized exclusively by microbes. These classes are sometimes referred to as pathogen associated molecular patterns (PAMPs) and include, for example, lipopolysaccharide (LPS), peptidoglycans, lipotechoic acids, and bacterial lipoproteins (BLPs).
LPS is an abundant outer cell-wall constituent from gram-negative bacteria that is recognized by the innate immune system. Although the chemical structure of LPS has been known for some time, the molecular basis of recognition of LPS by serum proteins and/or cells has only recently begun to be elucidated. In a series of recent reports, a family of receptors, referred to as Toll-like receptors (TLRs), have been linked to the potent innate immune response to LPS and other microbial components. All members of the TLR family are membrane proteins having a single transmembrane domain. The cytoplasmic domains are approximately 200 amino acids and share similarity with the cytoplasmic domain of the IL-1 receptor. The extracellular domains of the Toll family of proteins are relatively large (about 550-980 amino acids) and may contain multiple ligand-binding sites.
The importance of TLRs in the immune response to LPS has been specifically demonstrated for at least two Toll-like receptors, Tlr2 and Tlr4. For example, transfection studies with embryonic kidney cells revealed that human Tlr2 was sufficient to confer responsiveness to LPS (Yang et al., Nature 395:284-288 (1998); Kirschning et al. J Exp Med. 11:2091-97 (1998)). A strong response by LPS appeared to require both the LPS-binding protein (LBP) and CD14, which binds LPS with high affinity. Direct binding of LPS to Tlr2 was observed at a relatively low affinity, suggesting that accessory proteins may facilitate binding and/or activation of Tlr2 by LPS in vivo.
The importance of Tlr4 in the immune response to LPS was demonstrated in conjunction with positional cloning in lps mutant mouse strains. Two mutant alleles of the mouse lps gene have been identified, a semidominant allele that arose in the C3H/HeJ strain and a second, recessive allele that is present in the C57BL/10ScN and C57BL/10ScCr strains. Mice that are homozygous for mutant alleles of lps are sensitive to infection by Gram-negative bacteria and are resistant to LPS-induced septic shock. The lps locus from these strains was cloned and it was demonstrated that the mutations altered the mouse Tlr4 gene in both instances (Portorak et al., Science 282:2085-2088 (1998); Qureshi et al., J Exp Med 4:615-625 (1999)). It was concluded from these reports that Tlr4 was required for a response to LPS.
The biologically active endotoxic sub-structural moiety of LPS is lipid-A, a phosphorylated, multiply fatty-acid-acylated glucosamine disaccharide that serves to anchor the entire structure in the outer membrane of Gram-negative bacteria. We previously reported that the toxic effects of lipid A could be ameliorated by selective chemical modification of lipid A to produce monophosphoryl lipid A compounds (MPL(copyright) immunostimulant; Corixa Corporation; Seattle, Wash.). Methods of making and using MPL(copyright) immunostimulant and structurally like compounds in vaccine adjuvant and other applications have been described (see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094; 4,987,237; Johnson et al., J Med Chem 42:4640-4649 (1999); Ulrich and Myers, in Vaccine Design: The Subunit and Adjuvant Approach; Powell and Newman, Eds.; Plenum: New York, 495-524, 1995; the disclosures of which are incorporated herein by reference in their entireties). In particular, these and other references demonstrated that MPL(copyright) immunostimulant and related compounds had significant adjuvant activities when used in vaccine formulations with protein and carbohydrate antigens for enhancing humoral and/or cell-mediated immunity to the antigens.
Moreover, we have previously described a class of synthetic mono- and disaccharide mimetics of monophosphoryl lipid A, referred to as aminalkyl glucosaminide phosphates (AGPs), for example in U.S. Ser. Nos. 08/853,826, 09/074,720, 09/439,839 and in PCT/US98/09385, the disclosures of which are incorporated herein by reference in their entireties. Like monophosphoryl lipid A, these compounds have been demonstrated to retain significant adjuvant characteristics when formulated with antigens in vaccine compositions and, in addition, have similar or improved toxicity profiles when compared with monophosphoryl lipid A. A significant advantage offered by the AGPs is that they are readily producible on a commercial scale by synthetic means.
Although monophosphoryl lipid A and the AGPs have been described primarily for use in combination with antigens in vaccine formulations, their use as monotherapies, in the absence of antigen, for the prophyhlactic and/or therapeutic treatment of plant and animal diseases and conditions, such as infectious disease, autoimmunity and allergies, has not been previously reported.
The present invention, as a result of a growing understanding of certain mechanisms underlying the activities of monophosphoryl lipid A and AGP compounds, makes possible the novel therapeutic opportunities described herein.
In one aspect, the present invention provides methods for treating, ameliorating or substantially preventing a disease or condition in an animal by administering an effective amount of a compound having the formula: 
and pharmaceutically acceptable salts thereof, wherein X is xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94; R1 and R2 are each independently a (C2-C24)acyl group, including saturated, unsaturated and branched acyl groups; R3 is xe2x80x94H or xe2x80x94PO3R11R12, wherein R11 and R12 are each independently xe2x80x94H or (C1-C4)alkyl; R4 is xe2x80x94H, xe2x80x94CH3 or xe2x80x94PO3R13R14, wherein R13 and R14 are each independently selected from xe2x80x94H and (C1-C4)alkyl; and Y is a radical selected from the formulae: 
wherein the subscripts n, m, p and q are each independently an integer of from 0 to 6; R5 is a (C2-C24)acyl group (including, as above, saturated, unsaturated and branched acyl groups); R6 and R7 are independently selected from H and CH3; R8 and R9 are independently selected from H, OH, (C1-C4)alkoxy, xe2x80x94PO3H2, xe2x80x94OPO3H2, xe2x80x94SO3H, xe2x80x94OSO3H, xe2x80x94NR15R16xe2x80x94SR15, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CHO, xe2x80x94CO2R15, xe2x80x94CONR15R16, xe2x80x94PO3R15R16, xe2x80x94OPO3R15R16, xe2x80x94SO3R15 and xe2x80x94OSO3R15, wherein R15 and R16 are each independently selected from H and (C1-C4)alkyl; R10 is selected from H, CH3, xe2x80x94PO3H2, xcfx89-phosphonooxy(C2-C24)alkyl, and xcfx89-carboxy(C1-C24)alkyl; and Z is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94; with the proviso that when R3 is xe2x80x94PO3R11 R12, R4 is other than xe2x80x94PO3R13R14.
In certain illustrative aspects of the invention, the above methods are employed in treating, ameliorating or substantially preventing infectious diseases, autoimmune diseases and allergies.
The present invention, in other aspects, provides pharmaceutical compositions comprising one or more of the compounds described above in a suitable excipient, formulated and/or administered in the absence of exogenous antigen.